Guanine nucleotide exchange factor 2 for Rab5 proteins coordinated with GLUP6/GEF regulates the intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm.

Bottom Line:
The present study demonstrates that the double recessive type of glup4/rab5a and glup6/gef mutant showed not only elevated proglutelin levels and much larger paramural bodies but also reduced the number and size of PSVs, indicating a synergistic mutation effect.GEF proteins consist of the helical bundle (HB) domain at the N-terminus, Vps9 domain, and a C-terminal region.By the deletion analysis of GEFs, the HB domain was found essential for the activation of Rab5 proteins.

Mentions:
In order to elucidate the influence of the combined glup4/rab5a and glup6/gef mutations on intracellular transport of proglutelins, protein bodies in the endosperm of the single and double recessive type were analysed by immunofluorescence microscopy (Fig. 2, Supplementary Fig. S1). In the wild-type endosperm, protein bodies containing prolamines (protein body type I: PB-I) and those containing glutelins and α-globulins (protein storage vacuole: PSV) increased in size and number as the seed develops (Fig. 2A−C, Supplementary Fig. S1A−C). In each mutant type, prolamine containing PB-I were estimated to be similar in size and number to that seen for the wild type (Fig. 2B, E, H, K). In contrast, glutelin-containing PSVs were smaller and fewer in number in each mutant type (Fig. 2E, H, K) compared with the wild type (Fig. 2B). The severity of this condition varied among the mutant types, with glup4/rab5a showing a moderate reduction in size and number of PSVs (Fig. 2D, E) followed by glup6/gef (Fig. 2G, H) while the double recessive type showed the largest reduction in number of PSVs (Fig. 2J, K). Nearly all of the synthesized α-globulin was packaged in paramural bodies (PMBs) in the double recessive type (Fig. 2L).

Guanine nucleotide exchange factor 2 for Rab5 proteins coordinated with GLUP6/GEF regulates the intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm.

Mentions:
In order to elucidate the influence of the combined glup4/rab5a and glup6/gef mutations on intracellular transport of proglutelins, protein bodies in the endosperm of the single and double recessive type were analysed by immunofluorescence microscopy (Fig. 2, Supplementary Fig. S1). In the wild-type endosperm, protein bodies containing prolamines (protein body type I: PB-I) and those containing glutelins and α-globulins (protein storage vacuole: PSV) increased in size and number as the seed develops (Fig. 2A−C, Supplementary Fig. S1A−C). In each mutant type, prolamine containing PB-I were estimated to be similar in size and number to that seen for the wild type (Fig. 2B, E, H, K). In contrast, glutelin-containing PSVs were smaller and fewer in number in each mutant type (Fig. 2E, H, K) compared with the wild type (Fig. 2B). The severity of this condition varied among the mutant types, with glup4/rab5a showing a moderate reduction in size and number of PSVs (Fig. 2D, E) followed by glup6/gef (Fig. 2G, H) while the double recessive type showed the largest reduction in number of PSVs (Fig. 2J, K). Nearly all of the synthesized α-globulin was packaged in paramural bodies (PMBs) in the double recessive type (Fig. 2L).

Bottom Line:
The present study demonstrates that the double recessive type of glup4/rab5a and glup6/gef mutant showed not only elevated proglutelin levels and much larger paramural bodies but also reduced the number and size of PSVs, indicating a synergistic mutation effect.GEF proteins consist of the helical bundle (HB) domain at the N-terminus, Vps9 domain, and a C-terminal region.By the deletion analysis of GEFs, the HB domain was found essential for the activation of Rab5 proteins.